The long-term objective of our research is to understand the organization and expression of regulatory and structural gene systems in simple eukaryotic organisms. As a simple unicellular eukaryote, yeast is an excellent model system for studies on gene organization and regulation in higher cells. The methods we have developed over the past 3 years should now enable us to isolate and study any region of the yeast (or other simple eukaryote) genome, including centromeric DNA. Specific goals are: (1) Develop and exploit the yeast cloning systems, based on the use of cloned arg 4 and trp 1 DNAs on E. coli vectors, as vehicles for cloning yeast DNA in yeast cells; isolate key genes on chromosome III, such as pgk, cdc 10, alpha(mating-type locus), his 4. (2) Locate and define the centromeric DNA on chromosome III, in terms of restriction mapping and preliminary sequence analysis. (3) Investigate the mechanism of autonomous replication of the trp 1 and arg 4 plasmids in yeast cells. (4) Determine the deoxynucleotide sequences of a few representative Drosophila tRNA genes, including the surrounding control regions, (5) Further define the mechanism of yeast gene transcription and translation in E. coli and yeast cells; begin DNA sequencing studies on yeast gene control regions.